This application is a 371 of PCT/FR98/02727 filed Dec. 14, 1998.
Aids is induced by HIV-1 virus infection. Such virus consists in various proteins. Amongst such proteins, Tat is together with Nef protein produced the earliest in the viral cycle.
To fight against the effects of Tat protein various approaches have been proposed including genetic approaches to block Tat transcriptional action. Thus, clinical assays have taken place with antisense (Hybridon company), in vitro experiments have shown feasibility of ribozymes or mimics using the RNA of the TAR region (Tat binding site to mRNA).
Pharmacological molecules as inhibitors of Tat transcriptional effect have also been disclosed.
All these methods aim at blocking virus replication by blocking Tat transcriptional action.
Immunological methods have also been proposed for a long time.
For a specific immunization against native Tat protein, WO-A-91/18454 reports the use of polypeptides from retroviral proteins including native Tat protein, being obtained by proteolytic cleavages, chemical synthesis or genetic recombination, as immunogens to prevent the retroviral replication and the cytotoxic activity against in particular lymphocytes and nervous cells effected by retroviral proteins, in particular Tat. The toxicity of native proteins or the fragments thereof prevents however their use for an immunization.
WO-A-95/31999 reports the use of native Tat protein of HIV-1, fragments of said protein or polypeptides with deletions/substitutions as immunogens so as to block HIV-1 replication by blocking Tat extracellular protein capture by uninfected cells. In spite of the described immunogenicity, given the toxic effects of the native Tat protein in particular, the immune troubles it induces, and of the absence of neutralizing antibodies through immunization with the immunogens described therein, there is indeed a need to develop new inactivated Tat immunogens (which have lost all the nocuous effects from the native Tat protein) being adapted to induce a humoral and cellular immune response.
WO-A-96/27389 reports the use of new products, Tat toxoids and retroviral regulating proteins as immunogens adapted to induce an immune response against native Tat protein and prevent or amend the immunosuppressive effects thereof. Such toxoids, being inactive but immunogenic products, have been prepared by formaldehyde chemical treatment of the native protein or segments derived from such protein. However inactivation with aldehyde is uneasy. And for an industrial production regular results are necessary, mainly for inactivation. Moreover, Health agencies prefer products with a constant and well defined structure to authorize the sales thereof. An ideal product should further have a good stability over the time.
That is the reason why the present application aims at providing new toxoids and a simple and efficient new method to manufacture such toxoids, for example from HIV-1 regulating proteins which participate in the HIV-1 immunosuppressive effect or from alpha interferon the production of which is deranged by HIV-1 infection.
The toxoid strategy has great advantages with respect to aminoacid substitutions/deletions: the regions of native Tat protein which are responsible for the Tat pleomorphic effects are not well identified and it is difficult to predict a priori the substitutions/deletions which provide a total innocuity for the preparation thereof. For example mutation of C25 residue in G cancels Tat transactivating effect without suppressing the immunosuppressive effect thereof. On the other side, substitutions/deletions may alter strongly the structure of the modified molecule with respect to the native molecule (linear and conformational antigens) and thus prevent the development of an efficient immune reaction against the native molecule. This last point is particularly important for generating neutralizing antibodies against native protein.
An object of the present application is thus to provide a viral regulation protein or a fragment of a viral regulation protein or alpha interferon or a fragment of alpha interferon, characterized in that it is carboxymethylated.
In preferred implementation conditions for the invention, the above-mentioned protein or fragment comes from HIV-1, HIV-2, HTLV-1 or HTLV-2 virus, particularly HIV-1 or HIV-2 virus.
In other preferred implementation conditions for the invention, the above-mentioned protein or fragment is a viral regulation protein or a fragment of a viral regulation protein.
In further preferred implementation conditions for the invention, the above-mentioned protein or fragment comes from Tat, particularly from HIV-1.
Another object of the present application is also to provide a process for producing a protein or a fragment such as defined here-above, characterized in that said viral regulation protein or said fragment is submitted to a carboxymethylation step to obtain the expected compound which is isolated.
According to the invention, the proteins or fragments which have the same behaviour as toxins on the immune system, either the overproduced IFNxcex1, Tat or Nef, for example are modified with the carboxymethylation. Such chemical modification leads to new proteins or fragments which are biologically inactive, but hold their immunogenicity. In other words, such proteins or fragments which have the same behaviour as toxins on the immune system are recognized by antibodies produced against the carboxymethylated proteins or fragments according to the invention. Such carboxymethylated proteins or fragments hold enough immunogenic properties to create antibodies neutralizing or blocking said native protein and simultaneously have lost at least 90%, preferably at least 95%, more preferably at least 99% of the toxic biological properties of said native protein, namely its well known usual biological properties.
The carboxymethylated immunogenic compound according to the invention may consist in the totality or a fragment of the protein, including regulation protein, and may comprise, as it is well known by the skilled man in the art, one or more modifications in the aminoacids of such protein or fragment such as deletions, substitutions, additions or finctionalizations such as acylating aminoacids, inasmuch such modifications stay in the above-mentioned limits (no toxicity, immunological characters). For example, generally substituting an isoleucine residue for a leucine residue does not modify such properties. The modifications should generally concern less than 30% aminoacids, preferably less than 20%, more preferably less than 10%.
A fragment may comprise from 8 to 110 aminoacids for example, preferably from 12 to 60 aminoacids, more preferably from 12 to 40 aminoacids. Such a fragment may also comprise on the C- or N-terminal side(s) from 1 to 5 supplementary aminoacids, i.e. different from the origin segment. A fragment should also comprise one cystein at least to allow for carboxymethylation.
Generally, as to the modifications, the homology or similarity between the modified immunogene and the protein or part of native protein as well as the dimensions of the immunogenic compound and the use procedures or coupling of the immunogenic compound according to the invention with an immunogenic protein such as the tetanus toxoid, it may be referred in particular to equivalent WO-A-86/06414 or EP-A-0,220,273 or further PCT/US.86/0083 1 the teaching of which is incorporated therein by reference.
The immunogenic compounds according to the invention derived from regulation proteins will be called sometimes below in the experimental part xe2x80x9cviral toxoidsxe2x80x9d.
In fact, as for the conventional bacterial toxoids, they are free from own toxicity, but are adapted to provoke an immunization per administration to a subject.
To verify that the native regulation protein is well recognized by antibodies raised against said modified regulation protein, it is possible for example to check immunologically through Elisa in the presence of specific antibodies, the building of antigen-antibody complexes, as it will be shown below in the experimental part.
To see if the immunogenic properties of the regulation protein have been sufficiently preserved to create antibodies neutralizing said native protein, it is possible for example to immunize mammals (rabbits, rats, mice) with an immunogenic compound according to the invention and check that the produced antibodies for neutralizing the toxic activities for the Tat.
To check if the modified regulation protein has lost at least 90% of its toxic biological properties, it is possible for example as to the Tat to study the effect of the inactivated Tat on the immunosuppression of T cells or the neoangiogenesis.
Inactivation of Tat regulation protein is checked for example by the xe2x80x9cTat Rescue Assayxe2x80x9d by using a non infectious Tat deficient HIV mutant cultured on cell line HL-1 the replication of which depends upon an exogenous supply of native Tat.
The immunogenic compound according to the invention may derive particularly from any one of regulation proteins of the HIV-1, HIV-2, HTLV-1 or HTLV-2 viruses, including Nef, Rev, preferably Tat, of the HIV-1 and HIV-2 viruses.
The Tax protein of HTLV-1 or HTLV-2 may also be mentioned.
An object of the present application is also to provide a process for producing a protein or a fragment such as defined above, characterized in that said viral regulation protein or said fragment is submitted to a carboxymethylation step to obtain the expected compound which is isolated if desired.
The carboxymethylation reaction allows to modify the thiol groups (sulfhydryl group) being present at the cystein residues of the aminoacids chain. Such groups react with iodoacetic acid or iodoacetamide by a S-carboxymethylation or S-carboxyamidomethylation reaction, respectively.
For example, Tat protein has 7 cysteins. Such cysteins participate in the building of inter- and intra-chain disulfide bridges and assist oligomer formation.
The reaction product is in any case a S-carboxymethylcysteinyl or S-carboxymethylamidocysteinyl residue.
The carboxymethylation reaction may also be carried out with other chemical agents like performic acid, 3-bromopropionic acid, ethyleneimine, (2-bromoethyl) trimethyl ammonium bromide, 2-bromoethane sulfonate, 1,3-propanesulfone, etc.
In preferred implementation conditions for the above-mentioned process, said starting protein or fragment is under the form fused with a marker (FP) when it is submitted to carboxymethylation.
The starting proteins or fragments of the process are known products described in the literature, for example for Tat from HIV-1 by Frankel A. D. et al. (Cellular uptake of the tat protein from human immunodeficiency virus, Cell, 1988, 55:1189-93) or for Nef from HIV-1 by Azad A. A et al. (Large-scale production and characterization of recombinant human immunodeficiency virus type 1 Nef, J. Gen. Virol. 1994, 75:651-55), or could be prepared conventionally.
Starting proteins or fragments above-described may be in particular prepared:
1) by synthesis through genetic engineering or by biochemical synthesis,
2) by purification.
Genetic engineering allows to purify the proteins produced by affinity chromatography by using for example antibodies raised against the protein or one of the fragments thereof; the protein fused with a marker (FP) which will be used as an anchor for an affinity column may be also synthetized.
In other preferred implementation conditions for the above-mentioned process, when the protein or fragment is fused with a marker (FP), it is submitted to:
a concentration step, for example by ultrafiltration,
a desalting step for example by gel filtration,
a treatment with cyanogen bromide or enterokinase to cleave the fusion protein and thus release the protein or fragment,
concentration and diafiltration steps,
a chromatography by cationic exchange,
a concentration step by ultrafiltration followed by an exclusive gel filtration.
The above-mentioned cyanogen bromide reaction allows to cleave the thioethers. The cyanogen bromide reaction on the polypeptide molecules is selective by carrying out a cleavage at the existing methionine residues. Such a reaction ends to the formation of two polypeptide fragments per methionine residue. Such a reaction may be advantageously coupled with the above-described carboxymethylation reaction, but it is not necessary for the inactivation.
In other preferred implementation conditions for the above-mentioned process, the expected protein or fragment coupled with a compound to allow its purification, for example with a peptide fragment containing various histidines, preferably in a continuous sequence of 4, 5, including 6 histidines or more to allow the fixation with a nickel column is prepared. Inasmuch the presence of such a compound does not induce toxicity and does not modify disadvantageously the immunogenicity of the protein or the fragment, it is not necessary to cleave it after purification. However, in these preferred implementation conditions, such compound is cleaved to eliminate it.
The compounds that are the objects of the present invention have very interesting pharmacological properties. They are biologically inactive with respect to the functions they perform usually such as the immunosuppressive effects, the transactivating effects of the HIV-1 promotor or the oxidative effects for Tat, the anti Vesicular Stomatitis Virus (VSV) effects on the MDBK system or the immunosuppressive effects for the IFN-xcex1.
They are immunogenic for mice, but also for human beings. They allow in particular the induction of a humoral immune response with neutralizing antibodies and a cellular immune response.
Such properties are illustrated below in the experimental part. They justify the use of the above-mentioned proteins or fragments as well as the addition salts thereof with pharmaceutically acceptable acids as a medicament.
That is the reason why the invention also aims at a carboxymethylated protein or fragment as described above, for its use in a therapeutic treatment method for the animal or human body, i.e. as a medicament.
The medicaments according the present invention find their use for example in the curative as well as preventive treatment of the harmful effects caused by an overproduction of IFN-xcex1 or a viral regulation protein, including a retrovirus HIV-1, HIV-2, HTLV-1 or HTLV-2.
The Tat and Nef proteins have quite well preserved regions. However, the case being, the same patient will be able to be immunized with toxoids prepared from varying strains, including to adapt itself to geographic variations of the epidemic.
The immunogenic compounds according to the invention may be used as follows:
A patient requiring such a treatment is administered with an immunogenic compound according to the present invention, for example subcutaneously or intramuscularly, in a sufficient quantity to be efficient therapeutically. The administered dose may range for example from 50 to 1000 xcexcg intramuscularly as a e/h emulsion once a month during three months, and then periodically depending upon the induced serum antibody rate, for example every 2-6 months.
As medicaments, the carboxymethylated proteins or fragments from above may be incorporated to pharmaceutical compositions adapted for oral and mainly parenteral administration.
A composition according to the invention may be administered through any conventional way being used in the vaccine field, in particular subcutaneously, intramuscularly, intravenously or orally. The administration may be carried out in a single dose or in a repeated dose one or more times after a certain period of time.
The invention has also as an object to provide pharmaceutical compositions comprising at least an above-mentioned compound as an active ingredient.
In such compositions, the active ingredient is advantageously present in physiologically efficient doses; said compositions contain for example an efficient immunogenic dose of at least an active ingredient as described above. The immunogenic compound may be packaged alone or in a mixture with a pharmaceutically acceptable excipient such as a builder.
Such pharmaceutical compositions may be for example liquid and present under the pharmaceutical forms currently used in human medicine for the vaccines, like for example the injectable preparations including as an emulsion; they may be prepared according to the usual methods. The active ingredient(s) may be incorporated to the excipients usually used in such pharmaceutical compositions, such as aqueous carriers, calcium phosphate, alum, . . .
The invention aims including at pharmaceutical compositions such as:
a) a pharmaceutical composition comprising as a preventive or curative agent a viral toxoid or a fragment or similar of a regulation protein, for example Tat, according to the invention,
b) a pharmaceutical composition comprising as a preventive or curative agent anti-Tat antibodies produced from organisms immunized against such protein or the fragments thereof F(abxe2x80x2)2 or Fab according to the invention.
The present invention aims thus at a process for preparing an above-mentioned composition, characterized in that the active ingredient(s) are mixed, according to known methods per se, with acceptable excipients, including pharmaceutically acceptable ones.
Moreover, the invention concerns a kit comprising a vaccine pharmaceutical composition which, besides the active ingredient (for example Tat toxoid or derivatives or anti-Tat antibodies thereof), may comprise a builder and/or another immunogene with anti-retrovirus properties.
Another immunogene may include for example the envelope protein GP 160, including the transmembrane glycoprotein GP 10 of HIV-1 or the known fragments thereof in the state of art, the Gag protein of the viral capsid or even the Pol protein.
The invention aims finally at the use of an above-mentioned carboxymethylated protein or fragment so as to obtain a medicament adapted for a use as an immunogene.